Abstract
E3 ubiquitin (UB) ligases are the ending modules of the E1-E2-E3 cascades that transfer UB to cellular proteins and regulate their biological functions. Identifying the substrates of an E3 holds the key to elucidate its role in cell regulation. Here, we construct an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in cancer and neurodevelopmental disorders. We use yeast cell surface display to engineer E6AP to exclusively transfer an affinity-tagged UB variant (xUB) to its substrate proteins. Proteomic identification of xUB-conjugated proteins in HEK293 cells affords 130 potential E6AP targets. Among them, we verify that MAPK1, CDK1, CDK4, PRMT5, β-catenin, and UbxD8 are directly ubiquitinated by E6AP in vitro and in the cell. Our work establishes OUT as an efficient platform to profile E3 substrates and reveal the cellular circuits mediated by the E3 enzymes.
| Original language | English (US) |
|---|---|
| Article number | 2232 |
| Journal | Nature communications |
| Volume | 8 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 1 2017 |
Funding
We thank Wade Harper, Jon M. Huibregtse, Linda Hicke, and K. Dane Wittrup for providing the gene constructs of Uba1, E6AP, Rsp5, and yeast display vector. This work is supported by grants provided from the National Institutes of Health (GM104498 to J. Y. and H.K.; CA112282 to H.K.), the National Science Foundation (1420193 and 1710460 to J.Y.), the Chicago Biomedical Consortium (Catalyst-026 to H.K. and J.Y., PDR-010 to X.L.), Project 985 startup grant (WF220417001 and WF114117001/004 to B. Z.), the Lynn Sage Breast Cancer Research Foundation, and the Department of Pharmacology at Northwestern University.
ASJC Scopus subject areas
- General Chemistry
- General Biochemistry, Genetics and Molecular Biology
- General Physics and Astronomy
